(1) Field of the Invention
This invention concerns programmable, electrophoretic notch filter systems and methods that are useful in the isolation, purification and fractionation of analytes in solution. The invention generally relates to the field of analyte isolation and purification, and more specifically relates to the tunable electrophoretic isolation, purification, and fractionation of analytes such as proteins, peptides, polypeptides, protein complexes, antibodies, DNA, RNA, or other biological materials prior to subsequent analysis using, for example, mass spectrometry, ELISA, Surface Plasmon Resonance, or other common detection methods known in the art.
(2) Description of the Art
The purification, isolation and fractionation of complex biological samples is a central process required throughout molecular and cellular biology, pharmaceutical research and development, and medical diagnostics and a host of other fields. For example, in the case of genomics and “next gen” genomic sequencing, DNA fragments must be separated by size and targeted size fragments isolated for the robust creation of DNA sequencing DNA libraries.
In the field of “discovery proteomics,” defined as the global identification and quantification of all the proteins found in a biological sample, samples must be reproducibly fractionated according to a known and predictable physiochemical characteristic so as to reduce the dynamic range and complexity of the sample and enable sensitive analysis using methods such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). The abundance range of known plasma proteins, for example, spans over 10 orders of magnitude. Taken together with the heterogeneity introduced by post-translational and post-transcriptional modifications, the detection of low abundance proteins in complex multi-analyte samples remains a tremendous challenge and limits the discovery and validation of analytes that may serve as novel biomarkers and/or drug targets and further limits the deconvolution of cellular and molecular pathways.
In the field of “targeted proteomics,” in which the aim is generally to quantify one or more target proteins found in a biological sample, such as human plasma, target protein(s) must be significantly enriched—often by a factor of a million or more—relative to other proteins found in the sample so as to increase signal to noise and permit sensitive quantification, often using LC-MS/MS. Other analytical techniques, such as affinity assays using for example antibodies, require purification either before or after affinity interaction in order to reduce non-specific interactions and permit more robust, sensitive, and reproducible measurements.
In pharmaceutical discovery and development efforts, antibodies and other biotherapeutics must be isolated and purified so that they can be characterized for quality, function, mechanism of action, etc. Further, in molecular biology and biotherapeutic characterization, it is often desirable to purify analytes that interact with a specific protein, compound, or other molecular entity to, for example, deconvolute the therapeutic target and better understand the biological pathway. In these cases, the analyte complex must be purified from the other components in the sample so as to permit sensitive analysis. For these and a multitude of other applications well known to those engaged in these and related fields, methods for the isolation, purification, and fractionation of analytes are required.
A wide variety of methods are known for purifying analytes, such as those that utilize affinity interaction, filtration, electrophoretic separation and chromatographic separation, including but not limited to reverse phase chromatography, gel permeation chromatography, size exclusion chromatography, and cation exchange chromatography.
Generally, when isolating, purifying and fractionating analytes, it is desirable that the system used to do so have a high load capacity, such that a large amount of total analyte can be loaded into the system. Since many target analytes are present at concentrations that are millions of times less than the concentration of the most abundant molecules in the sample, a high load capacity ideally permits preparation of enough sample so as to permit detection of low abundance molecules.
Second, it is usually desirable to provide for the isolation, purification, and fractionation of analytes in a manner that is highly specific and has high resolution. In other words, the preparation system preferably provides for the extraction of the target analyte in as ‘neat’ of a sample as possible; i.e. ideally only one type of protein is found in a single isolated fraction such that it's signal to noise is optimized.
A further desire is that the method for isolation, purification, and fractionation is highly reproducible, so that the same system and method can be used repeatedly on multiple samples without introducing variability, thereby providing clear and indisputable results. It is also a desire that the methods and system provide for high recovery of, and reproducibility of recovery of, the analytes that have been isolated, purified, and fractionated.
Furthermore, it is desirable that the methods for isolating and purifying analytes be versatile, such that they can be easily adapted to other analytes and are generally useful. For example, methods such as size exclusion chromatography are generally useful for purifying a large range of analytes following some initial method development for each target analyte and may be used to purify multiple analytes or fractions from a single sample in sequential fashion. However, these methods may suffer from relatively poor analyte recovery and poor specificity/resolution. Conversely, affinity purification using a highly specific antibody provides relatively high enrichment, but requires the production of a specific antibody for each analyte targeted for purification.
It is further desirable that any single isolation or purification system/method be compatible with other methods/systems for isolation, purification and fractionation, as multiple dimensions of separation/enrichment are often required. Lastly, it is desirable that such systems and methods be easy to use and easy to optimize for various samples and target analytes.
Perhaps the most widely used method for separating, purifying and isolating charged analytes is one dimensional electrophoresis (1DE), typically using either agarose or polyacrylamide gel electrophoresis (PAGE). 1DE permits the separation of analytes, such as DNA, proteins, peptides, polypeptides, protein complexes, RNA, etc. on the basis of electrophoretic mobility. Under an applied electric field, charged analytes migrate through and are sieved by polymeric gel matrix. As such, analytes with a high mass to charge ratio move more slowly than analytes with a low mass to charge ratio and are thus separated.
Used together with sodium dodecyl sulfate (SDS), a uniform net charge is imparted on all proteins in a sample such that the separation accomplished strictly on the basis of molecular weight, rather than on the basis of mass-to-charge. A multitude of other 1DE methods are well known, including but not limited to clear native PAGE, blue native PAGE, field inversion electrophoresis, counter current electrophoresis, capillary electrophoresis and the like. 1DE may be practiced in a slab gel format, in a tube gel format, or some combination of symmetric or asymmetric formats, as has been well published. As such, 1DE is one of the most widely practiced and generally useful techniques for analyte isolation, purification, and fractionation owing to its ability to provide separation on the basis of electrophoretic mobility or molecular weight, resolving power over a large mass range, ease of use, and versatility.
During the 1DE process, the electric field is typically applied as a constant voltage for a continuous period of time sufficient to allow for complete separation of the sample analytes across the entire length of the gel matrix. The voltage is discontinued only at the end of the experiment, immediately before the most electrophoretically mobile analytes reach the end of the gel. The gels can then be imaged, using fluorescence, dyes or other methods well known in the art, and the location of the separated analyte ‘bands’ detected. Following detection, one or more bands in the gel may be cut out using a razor blade or other similar tool.
A number of methods are known for then recovering the purified analytes from the extracted gel band. The most widely used methods for gel plug purification rely upon enzymatic or other chemical cleavage of the analytes into smaller components that are diffused from the gel plug pores into a surrounding liquid. This method, while useful, is limited in that large, intact analytes, e.g. proteins, protein complexes, etc., must be cleaved into smaller subcomponents. Thus, direct analysis of the intact analyte is generally difficult, if not impossible. In certain applications involving the analysis of proteins in particular, this limitation prevents the facile analysis of post-translational modifications, truncations, splice variants and other features necessary to fully characterize the gene products in the sample. Additionally, the method is highly labor-intensive and tedious and recovery from the gel plugs is relatively low, estimated to be between 15-60%, severely limiting downstream analysis. These limitations severely limit the method's utility in a range of important applications.
An alternative method of analyte recovery from 1DE involves electro-elution of analytes from the extracted gel plug into a solution or onto a membrane. In this case, the cut gel plug is placed into a chamber and an electric field is directed through the gel plug so as to electrophoretically move the target analyte from the gel and into liquid or a capture support (e.g. capture membrane, reverse phase surface, etc.). While useful for some applications, the electro-elution technique is generally inefficient and unable to recover large molecules with high recovery, as has been well published. The technique is also labor-and-time intensive, since it involves the manual extraction of gel plugs and insertion of those plugs into a second apparatus and running of a second apparatus. The method also often results in excessive analyte dilution.
A third method involves the continuous elution of separated analytes from the end of the gel, most often a tube gel, under an applied electrophoretic field into a flowing stream of liquid. In this mode, analytes are separated on the basis of their electrophoretic mobility through the gel. Rather than discontinuing the voltage before the higher mobility analytes reach the end of the gel and cutting the separated bands from the gel matrix, the voltage is continued so as to elute the separated analytes from the gel matrix. As such, the voltage is applied continuously until the last to elute analytes are eluted from the gel matrix and swept into the collecting liquid flowing at the end of the gel matrix. A robotic fraction collector may be used to move the eluate from one collection vial to another, thereby permitting ‘fractionation’ of the sample.
This method offers several advantages over other 1DE methods for analyte isolation, purification, and fractionation, chief among them the ability to recovery separated analytes intact, in solution. However, limitations of the continuous elution gel electrophoresis method include usability, reproducibility, and dilution. Since analytes are eluted from the separation medium into a flowing stream of liquid, those analytes that elute from the gel at a slower rate and thus require longer to fully elute from the gel are continuously diluted, which is highly undesirable. Furthermore, since voltage must be applied continuously using these systems and methods, there is no effective method for capturing specific, predetermined analyte fractions in a multiplexed, high throughput format, which is highly desirable. Furthermore, no existing system permits the use of ready-to-use kits that have been manufactured in a consistent, reproducible fashion and which permit consistent results run-to-run. Since analytes are eluted from the gel matrix, rather than ‘developed’ in a standard 1DE method, a respective analyte's migration time must be exactly the same run-to-run if that analyte is to be isolated and purified in a reproducible manner.
It is thus an object of this invention to overcome the various limitations of the prior art discussed above and to describe a system and methods for the improved isolation, purification, and fractionation of analytes.